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The Effect of Mechanical Force on Gene Expression of Human Bladder Smooth Muscle Cells" (2012)

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The Effect of Mechanical Force on Gene Expression of Human Bladder Smooth Muscle Cells Philadelphia College of Osteopathic Medicine DigitalCommons@PCOM PCOM Biomedical Studies Student Scholarship Student Dissertations, Theses and Papers 6-2012 The ffecE t of Mechanical Force on Gene Expression of Human Bladder Smooth Muscle Cells Christopher A. Callan Philadelphia College of Osteopathic Medicine, [email protected] Follow this and additional works at: http://digitalcommons.pcom.edu/biomed Part of the Molecular Genetics Commons, and the Urogenital System Commons Recommended Citation Callan, Christopher A., "The Effect of Mechanical Force on Gene Expression of Human Bladder Smooth Muscle Cells" (2012). PCOM Biomedical Studies Student Scholarship. Paper 36. This Thesis is brought to you for free and open access by the Student Dissertations, Theses and Papers at DigitalCommons@PCOM. It has been accepted for inclusion in PCOM Biomedical Studies Student Scholarship by an authorized administrator of DigitalCommons@PCOM. For more information, please contact [email protected]. PHILADELPHIA COLLEGE OF OSTEOPATHIC MEDICINE Philadelphia, Pennsylvania The Effect Of Mechanical Force on Gene Expression of Human Bladder Smooth Muscle Cells A thesis submitted in partial fulfillnment of the requirements for the degree of MASTER OF BIOMEDICAL SCIENCE by Christopher A. Callan June 2012 We approve the thesis of Christopher A. Callan _______________________________________________________________________ Edward J. Macarak, Ph.D. Date Chairman; Department of Anatomy and Cell Biology University of Pennsylvania School of Dental Medicine Thesis Advisor _______________________________________________________________________ Pamela S. Howard, Ph.D Date Professor; Department of Anatomy and Cell Biology University of Pennsylvania School of Dental Medicine _______________________________________________________________________ Christopher S. Adams, Ph.D. Date Professor; Department of Anatomy Philadelphia College of Osteopathic Medicine _______________________________________________________________________ Wenjie Wei, MD Date Assistant Professor; Department of Anatomy and Cell Biology University of Pennsylvania School of Dental Medicine Abstract Cells are able to sense their physical surroundings through mechanotransduction. In doing so, they can translate the mechanical forces and deformations into a wide range of biochemical signals and genetic programs which can adjust their structure and extracellular environment. The urinary bladder is a physically active organ that undergoes periodic stretching as part of its normal function. In the bladder, abnormal pressure from a variety of pathologic conditions can result in forces which, over time, can alter the genetic expression of the proteins of the extracellular matrix. To determine the role that stretching or mechanical deformation may play in altering the synthetic phenotype of bladder wall cells, a series of experiments were carried out to quantify several extracellular matrix messenger ribonucleic acids (mRNAs) and their corresponding protein levels after mechanical challenge. This report first summarizes the current knowledge about regulation of cell function by mechanical forces and then presents data on the effect(s) of mechanical force on several mechanosensitive genes expressed by human bladder smooth muscle cells (BSMCs). Using this experimental system, we demonstrated that BSMCs were acutely sensitive to mechanical deformation and showed alteration of many structural and extracellular matrix genes. This study demonstrates that BSMC extracellular matrix secretory phenotype can be altered by the mechanical deformation experienced by these cells. These data support the concept that deformation of the bladder wall affects the secretory phenotype of BSMCs and can result in an altered extracellular matrix composition. iii TABLE OF CONTENTS 1. INTRODUCTION 1.1 SIGNIFICANCE………………………………………………………1 1.2 BACKGROUND……………………………………………………...2 1.3 PROPOSAL…………………………………………………………...5 2. DETAILED METHODS 2.1 TENSORCELL STRAIN…………………………………………….18 2.2 MEMBRANE-MATRIX ENGINEERING………………………….20 2.3 ACQUISITION AND GROWTH OF HUMAN BLADDER SMOOTH MUSCLE CELLS……………………...21 2.4 PROTOCOL FOR SUBCULTIVATION OF HUMAN BLADDER SMOOTH MUSCLE CELLS………………………21 2.5 PROTEIN LOCALIZATION/ IDENTIFICATION…………………22 2.6 HUMAN BLADDER SMOOTH MUSCLE CELL PLATING TO TENSORCELL WELLS………………………...24 2.7 APPLICATION OF STRAIN TO HUMAN BLADDER SMOOTH MUSCLE CELLS……………………………………24 2.8 PURIFICATION OF TOTAL RNA FROM HUMAN BLADDER SMOOTH MUSCLE CELLS………………………24 2.9 REVERSE TRANSCRIPTION (RT) OF TOTAL RNA FROM HUMAN BLADDER SMOOTH MUSCLE CELLS TO COMPLIMENT DNA (cDNA)……………………..26 2.10 QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (PCR) OF cDNA………………………….27 3. EXPERIMENTAL EVALUATION 3.1 IMMUNOLOCALIZATION OF STRETCH- ASSOCIATED PROTEINS IN HUMAN BLADDER SMOOTH MUSCLE CELLS……………………………………30 3.2 QUANTITATIVE RT-PCR ANALYSIS……………………………31 3.3 DISCUSSION………………………………………………………..44 4. REFERENCES iv LIST OF FIGURES Figure 1 Dystrophin-Glycoprotein Complex……………………….....................13 Figure 2 TensorCell Stretch Apparatus……………………………………....…...18 Figure 3 2% Gel Electrophoresis of Control and Experimental RNA…………….26 Figure 4 TaqMan 96-well PCR Plate…………………………………………...…28 Figure 5 Immunohistochemistry Analysis of β-Sarc & α-1 [III] Collagen...……..30 Figure 6 Immunohistochemistry Analysis of α-, β-, γ-, δ-sarcoglycans………..…30 Figure 7 Immunohistochemistry Analysis of Desmin………………………....….31 Figure 8 Immunohistochemistry Analysis of Vimentin……………………...…...31 Figure 9 RNA Concentration of Experimental Samples…………………...……..32 Figure 10 Quantitative PCR Analysis of α-1 [III] Collagen…………………….....33 Figure 11 Quantitative PCR Analysis of Connective Tissue Growth Factor……...33 Figure 12 Quantitative PCR Analysis of β-Integrin……….………………….....…34 Figure 13 Quantitative PCR Analysis of Paxillin…………………………………..34 Figure 14 Quantitative PCR Analysis of Desmin…………………………………..34 Figure 15 Quantitative PCR Analysis of Vimentin……………………….......……34 Figure 16 Quantitative PCR Analysis of Matrix Metalloprotease-1………………..35 Figure 17 Quantitative PCR Analysis of Tissue Inhibitor of Metalloprotease-1…...35 Figure 18 Quantitative PCR Analysis of α-Sarcoglycans…………………………..35 Figure 19 Quantitative PCR Analysis of β-Sarcoglycans…………………………..35 Figure 20 Quantitative PCR Analysis of δ-Sarcoglycans…………………………..36 Figure 21 Quantitative PCR Analysis of ε-Sarcoglycans………………….……….36 Figure 22 Quantitative PCR Analysis of γ-Sarcoglycans…………………………..36 Figure 23 Quantitative PCR Analysis of ζ-Sarcoglycans…………………………..36 v LIST OF TABLES Table 1 Volume of Buffer RLT to Tissue Disruption Homogenization…….……25 Table 2 Volume of Kit Components Used for Each Experiment………….…..…27 Table 3 Optimized Conditions for RNA-to-cDNA Kit……………...…..……….27 vi ACKNOWLEDGEMENTS This thesis would not have been possible without the guidance and help of several individuals who in one way or another contributed and extended their valuable assistance in the preparation and completion of this study. First and foremost, my utmost gratitude to Dr. Edward J. Macarak, Chairman of the Anatomy and Cell Biology Department at the University of Pennsylvania School of Dental Medicine and chair of my research committee, who sincerity and encouragement I will never forget. Dr. Macarak has been my inspiration during my research as well as my greatest teacher. Dr. Pamela S. Howard, Professor of Anatomy and Cell Biology at the University of Pennsylvania School of Dental Medicine, who provided limitless advice patience throughout my studies and was pivotal in the completion of my research. Dr. Christopher S. Adams, Professor of Anatomy at the Philadelphia College of Osteopathic Medicine, who gave excellent guidance and instruction throughout my project and was always willing to provide feedback when needed. Dr. Wenjie Wei for his input which I found to be among the most helpful advice I have received throughout my research. The administrators and faculty of both the Philadelphia College of Osteopathic Medicine and the University of Pennsylvania School of Dental Medicine, for their untiring efforts of encouragement and support on my behalf. Finally, and most importantly, to Robert Peter Callan, my father and greatest inspiration. His name has never before been written on academic literature, nor have any of his achievements been printed in scholarly or scientific journals; yet I happily remain in the shadow of his success forever. vii 1. INTRODUCTION: The purpose of this project is to define, at the molecular level, the process by which gene expression of the extracellular matrix is regulated by mechanical forces in the Human Bladder Smooth Muscle cells (BSMCs). The goal is to first localize several functionally distinct transmembrane proteins; Sarcoglycans (α, β, γ, δ and ε), cytoskeletal proteins Vimentin, and Desmin to verify their presence in the cultured BSMCs using fluorescent-labeled antibodies specific for each protein. The sarcoglycans are primarily responsible for transferring intracellular force generated by the interaction of actin and myosin while the extracellular proteins are responsible for linking the cells to the extracellular matrix. These proteins comprise several collagen matrix proteins, e.g., type I, type III and type IV collagens and an integrin-receptor (11, 64). To evaluate the functions of these proteins, we will subject the BSMCs, using a cell strain apparatus, to a level of force which mimics those experienced by the bladder wall during the filling/emptying cycles. This investigation
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